Vol. 46, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1983, p. 348-3550099-2240/83/080348-08$02.00/0Copyright © 1983, American Society for Microbiology

Effect of H2-CO2 on Methanogenesis from Acetate orMethanol in Methanosarcina spp.THOMAS J. FERGUSON AND ROBERT A. MAH*

Division ofEnvironmental and Nutritional Sciences, School ofPublic Health, University of California, LosAngeles, California 90024

Received 7 March 1983/Accepted 27 May 1983

Growth of Methanosarcina sp. strain 227 and Methanosarcina mazei on H2-CO2 and mixtures of H2-CO2 and acetate or methanol was examined. The growthyield of strain 227 on H2-CO2 in complex medium was 8.4 mg/mmol of methaneproduced. Growth in defined medium was characteristically slower, and cellyields were proportionately lower. Labeling studies confirmed that CO2 wasrapidly reduced to CH4 in the presence of H2, and little acetate was used formethanogenesis until H2 was exhausted. This resulted in a biphasic pattern ofgrowth similar to that reported for strain 227 grown on methanol-acetate mixtures.Biphasic growth was not observed in cultures on mixtures of H2-CO2 andmethanol, and less methanol oxidation occurred in the presence of H2. In M.mazei the aceticlastic reaction was also inhibited by the added H2, but since thecultures did not immediately metabolize H2, the duration of the inhibition wasmuch longer.

Radioactive isotopic labeling studies indicateat least two main substrates for methane produc-tion from nonmarine fermentations in nature: (i)CO2 reduction by H2 to CH4 and H2O, and (ii)acetate splitting with reduction of the methylgroup and oxidation of the carboxyl group toform CH4 and CO2, respectively (3, 5, 15, 18-21). In mixed methanogenic ecosystems (i.e.,anaerobic digesters), both types of substratesare continuously turning over. Although H2 isnot detectable at substrate concentrations be-cause of its rapid turnover, it accounts for ca.30% of the methane formed in digester fermenta-tions (12). It is also now well appreciated as anintermediate in oxidation-reduction reactions in-volving interspecies H2 transfer. Its potentialrole in regulating other methanogenic substrateshas not been reported, although it accumulateswhen methane formation is inhibited in anaero-bic digesters or has not yet occurred in batchenrichment cultures.Methanosarcina sp. strain 227 may utilize H2-

CO2, acetate, methanol (10), or trimethylamine(unpublished finding) for methanogenesis; how-ever, it preferentially metabolizes methanol be-fore acetate in mixtures of methanol and acetate(18). Trimethylamine was metabolized beforeacetate by Methanosarcina barkeri strain Fu-saro grown on mixtures of these two substrates(4). Evidence is presented that methanogenesisfrom acetate was limited in the presence ofadded H2, but methanogenesis from methanolwas not similarly affected. In Methanosarcina

mazei, methanogenesis from acetate ceased inthe presence of added H2, and methanogenesisfrom H2-CO2 did not immediately occur. Aninhibitory effect of H2 (7, 10, 13), trimethylamine(4), or methanol (19) on the aceticlastic reactionin Methanosarcina spp. was previously report-ed. This inhibition was not entirely supported byScherer and Sahm (17) or Hutten et al. (7).Methanol and acetate were reportedly usedsimultaneously (17), whereas H2 inhibited ace-tate utilization only in the absence of CO2 (7).We present new information on the effect of H2-CO2 on acetate and methanol utilization inMethanosarcina spp. and reconcile these appar-ent differences.

(Portions of these results were previouslyreported [T. J. Ferguson and R. A. Mah, Abstr.Annu. Meet. Am. Soc. Microbiol. 1979, I78, p.108].)

MATERIALS AND METHODSBacterial strains. Methanosarcina sp. strain 227 and

M. mazei (S-6), formerly Methanococcus mazei (R. A.Mah and D. A. Kuhn, submitted for publication), weremaintained in stock culture as previously described (9,10).

Culture media. Two types of mineral media wereused for growth yield determinations. Medium 1 (car-bonate buffered) contained the following (per liter oftap water): NH4CI, 1.0 g; K2HPO4 3 H20, 0.4 g;MgCl2 * 6 H20, 0.1 g; 0.10%o resazurin, 1.0 ml;NaHCO3, 1.0 g. Although the media in our studieswere first made up in tap water, distilled water waslater substituted and adopted without any change in

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METHANOSARCINA METABOLISM OF MIXED SUBSTRATES 349

our findings. The medium was prepared and dispensedin 50- to 100-ml portions into 250-ml boiling flasks orserum vials (1) under 02-free N2-CO2 (70:30) andstoppered with butyl rubber stoppers that were wiredin place. Media were autoclaved at 120°C and 15 lb/in2for 20 min. Before inoculation, 3.0 ml of a sterile,anaerobic solution of 1% Na2S and 5% Na2CO3 wasadded to each flask. Mineral medium 2 (phosphatebuffered) consisted of the same inorganic salts asmedium 1, with the addition of 10.7 g of Na2PO4 * 7H20 and the deletion of NaHCO3. The medium wasprepared and dispensed under O2-free N2 (6). Beforeinoculation, 3.0 ml of 1% Na2S per 100 ml of mediumwas added to each flask. The final pH of both mineralmedia was 6.9 to 7.1.The complex medium for growth yield and substrate

utilization studies was the 0.2% yeast extract andTrypticase (BBL Microbiology Systems, Cockeys-ville, Md.) medium (with sodium bicarbonate) de-scribed previously (18) except that distilled water wasused in place of tap water. Acetate (0.05 to 0.10 M),methanol (0.10 M), or H2-CO2 (70:30; 250 KPa) wereadded as growth substrates where noted.

Culture conditions. Cultures were maintained bytransfer at 3- to 5-day intervals for cells on complexH2-CO2 or methanol media, 5- to 7-day intervals forthe defined H7-CO2 medium, and 7- to 10-day inter-vals for the complex acetate medium. Inocula of 1 to2% were used. Stock cultures were grown on acetateor methanol at 37°C without shaking. Stock cultureson H2-CO2 and all cultures used in the substrate andgrowth studies were shaken at 37°C in a reciprocating-style water bath (model 2156; Research Specialties,Inc., Richmond, Calif.) operated at speed setting 6(120 strokes per min).

Analysis of gases. Methane and carbon dioxide wereanalyzed as previously described (2). Hydrogen wasdetermined by using a Loenco model 2000 gas chro-matograph equipped with a thermal conductivity de-tector (60 mA) and a 2-ft by 0.25-in. (ca. 60.96- by 0.63-cm) aluminum column packed with 80-100 meshPoropak Q (Applied Sciences Laboratories, State Col-lege, Pa.). The carrier gas was 100%o N2 (60 ml/min),and the injector, column, and detector were operatedat 30°C for 0.10- to 0.50-ml samples.

Ceil growth yields. Cell yields were determined nearthe peak of exponential growth for 100-ml culturevolumes of strain 227 grown on H2-CO2. Typically theflasks were regassed twice before completion of theexperiment. Cells were harvested by use of a clinicalcentrifuge, collected on membrane filters (Gelman 47

TABLE 1. Summary of growth yield data onMethanosarcina sp. strain 227 grown in H2-CO2

Yield (mg/mmol) No. ofMediuma determi- SD

Range Mean nations

Complex medium 6.71-10.35 8.38 20 0.88Mineral medium 1 5.3-9.5 6.4 7 1.6

(carbonate buffer)Mineral medium 2 2.79-7.90 5.40 7 1.87

(phosphate buffer) I_a See the text for the composition of medium 1 and

medium 2.

mm, 0.45-,um pore size), and dried to constant weightwith a vacuum oven at 60 to 70°C as previouslydescribed (19).

Labeling studies. Sterile, anaerobic, radioactive sub-strates were prepared in culture tubes as previouslydescribed (18). Radioactive substrates were added toyield media concentrations of 0.01 to 0.10 pCi/ml.Radioactivity in the gaseous fermentation productswas determined by the method of Baresi et al. (2) byassaying the headspace. Dissolved gases were calcu-lated from Henry's law (28).

Isotopes. Sodium [2-14C]acetate (specific activity, 98mCi/mmol); sodium [14C]carbonate (specific activity,53 mCi/mmol); and [14C]methanol (specific activity,3.9 mCi/mmol) were purchased from Schwartz/Mann,Orangeburg, N.Y.

RESULTSGrowth studies. Methanosarcina sp. strain 227

did not exhibit a lag in growth or methanogene-sis when transferred from acetate to H2-CO2 incomplex medium; it had a doubling time of ca. 9h. However, growth of strain 227 on H2-C02 inmineral medium required prior growth on H2-CO2 in complex medium containing yeast ex-tract and Trypticase. Once growth on H2-CO2was initiated, strain 227 could be transferred andmaintained on mineral medium. The results ofseveral growth yield determinations for bothmineral and complex media are summarized inTable 1. Growth on H2-CO2 in mineral mediumwas characteristically slower than in complexmedium, with doubling times between 12 and 24h. The average growth yields were also consid-erably lower, with greater variations in yields.M. mazei did not grow well enough on H2-CO2in complex or mineral medium to obtain anygrowth yield data.

Substrate utilization. Radioactive labeling ex-periments were performed with cells pregrownon complex acetate medium and inoculated intocomplex acetate medium containing either 100%N2 or an H2-CO2 (70:30) atmosphere. The fol-lowing results were obtained for Methanosar-cina sp. strain 227. (i) H2-CO2 was used immedi-ately as a methanogenic substrate when cellswere pregrown on acetate. Once H2 was ex-hausted, the reduction of CO2 ceased. (ii) Meth-anogenesis from acetate lagged in the presenceof H2-CO2, but once H2 was exhausted, methan-ogenesis from acetate was rapid and complete.(iii) In the absence of H2-CO2, virtually no CO2was reduced to methane, and methanogenesisfrom acetate did not lag. Similar results wereobtained whether the flasks contained an atmo-sphere of 100% N2 or N2-CO2 (70:30).These results indicated that the utilization of

acetate might be regulated in response to H2concentration. A more refined time course studywas initiated to examine methanogenesis fromacetate in the presence of H2. Strain 227 was

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350 FERGUSON AND MAH

0

0

E

c0

a0

:=

C

o£c

aA*0

..aftf0Scx

.Coo

0a

105 L1-

Time (hours)FIG. 1. Methanosarcina sp. strain 227 pregrown in

complex medium with 0.05 M sodium acetate. Imme-diately after inoculation of fresh medium the flaskswere gassed with H,-CO2 (70:30; 250 KPa). Themedium contained 100 ml of complex medium, 0.05 Msodium acetate, and 10 ,Ci 14CH3COO0). Symbols: *,H2; *, CH4; 0, 14CH4.

pregrown on complex acetate medium and in-oculated into fresh complex acetate mediumcontaining [2-14C]acetate and H2-C02 (Fig. 1).During the first 90 h, methanogenesis, presum-ably from H2-CO2, proceeded exponentially.When H2 was exhausted, between 98 and 99 h,less than 1% of the acetate initially present wasconverted to methane via the aceticlastic reac-tion. After 99 h the aceticlastic reaction re-sumed, and all methane was derived from ace-tate.These results (Fig. 1) confirmed the existence

of a lag period for the metabolism of acetate tomethane in the presence of H2-C02. To examinethe effect of H2 on a large aceticlastic populationof strain 227, we grew the Methanosarcina sp. inacetate medium and then pressurized the flaskswith H2-C02 (Fig. 2). The addition of H2-CO2inhibited methanogenesis from acetate. H2 wasexhausted rapidly, and methanogenesis fromacetate then resumed. When H2 was replenished(data not shown), inhibition of methanogenesisfrom acetate continued. In control cultures pres-surized under N2-CO2, methanogenesis was un-affected (data not shown).Such phenomena were not observed for all

aceticlastic methanogens. In M. mazei, for ex-ample, acetate was not metabolized in the pres-ence of H2, and H2 itself was not oxidizedthroughout the 12-day experimental period (Fig.

3). The addition of N2-CO2 had no effect on theaceticlastic reaction in M. mazei (Fig. 4). Zinderand Mah (28) reported a similar finding in Meth-anosarcina sp. strain TM1, a thermophilic aceti-clastic methanogen that did not metabolize H2.

Cultures of strain 227 pregrown on H2-C02for several transfers were also examined withradioactively labeled substrates. In complex ac-etate medium containing H2-C02, very little [2-14C]acetate was converted to 14CH4 before thedisappearance of H2. However, once H2 wasdepleted, methanogenesis from acetate was im-mediate and as rapid as in cultures pregrown onacetate. When cells pregrown on H2-CO2 weretransferred to complex medium containing ace-tate as the sole methanogenic substrate, agrowth lag of 4 to 8 weeks occurred. During thislag there was no visible change in culture turbid-ity, and little methane was produced. Whengrowth finally occurred, it resembled that ofacetate-grown cultures.The formation of 14CH4 and 14CO2 by cells

pregrown on acetate, methanol, or H2-C02 wasdetermined by assaying the headspace gases ofcultures acidified with HCl after growth andmethanogenesis had ceased (Tables 2 and 3). InTable 2, the final amount of 14CH4 producedfrom [2-14C]acetate by cells pregrown on acetatewas similar regardless of the presence or ab-sence of H2. This indicated that the quantity ofacetate ultimately catabolized to CH4 was simi-lar under these two conditions. However, the

a0

Ea.

3sc0a.5laC4S

S6U

'U-G.

ca

I aC

qe

Time (days)FIG. 2. Methanosarcina sp. strain 227 growing in

complex 0.05 M sodium acetate medium with 5 ,Ci of14CH3COO, gassed with H2-CO2 (70:30; 250 KPa) asindicated by the arrow. Symbols: *, H2; 0, CH4; O,14CH4.

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METHANOSARCINA METABOLISM OF MIXED SUBSTRATES 351

E0x' 103 _ / 106c

ILa~0

d02 VS

lo, g 1042 6 10 14 is 2

Time (days)FIG. 3. M. mazei growing in complex 0.05 M sodi-

um acetate medium with S ,uCi of 14CH3C00-, gassedwith HZ-CO2 (70:30; 250 KPa) as indicated by thealTow. Symbols: *, H2; *, CH4; El, 14CH4.

amount of A4C02 produced from [2-14C]acetatetended to be greater in the presence than in theabsence of H2. This showed that the methylgroup of acetate may be oxidized to a greaterextent when H2 is available. The labeling of14CH4 in the presence of 14CO2 and H2 reflectedthe expected reduction of C02 by H2. In metha-nol-grown cultures containing both methanoland H2-CO2 (Table 3), less H2 was used toreduce C02 to CH4 when methanol was present,and less methanol was oxidized when H2 was

present. By calculation based on the quantity of4c02, the quantity of methane produced byoxidation and reduction of methanol (reaction 2,Table 4) accounted for approximately 53% of theradioactive methane formed in experiment 2,Table 3. The remainder of the radioactive meth-ane (ca. 47%) was produced by reduction of14CH30H by H2. Likewise, the oxidation of H2with Co2 as the final electron acceptor wasdecreased by approximately7rio by the pres-ence of methanol (Table 3). Presumably, the H2was diverted to methanol reduction.

DISCUSSIONThe ability of Methanosarcina sp. strain 227

to metabolize acetate was influenced by thechoice of substrate for pregrowth of cells. Strain227 grew well on H2-C02 when pregrown onacetate or methanol. Transfer of growing cellsfrom H2CO2 to acetate medium, however, re-sulted in an extended growth lag. The transfer

from H2-CO2 to acetate in complex medium wasfacilitated when the H2-CO2-grown cells werefirst transferred to a complex medium containingboth H2-CO2 and acetate. Thus, pregrowth ofMethanosarcina sp. strain 227 on either H2-CO2or methanol for several transfers may select forpopulations incapable of immediate acetate utili-zation. Growth lags varying between 4 and 8weeks were observed for strain 227 pregrown onH2-CO2 and transferred to acetate medium. Thisphenomenon may be responsible for difficultiesreported in culturing M. barkeri strain MS onacetate as the sole methanogenic substrate (25,27). A similar lag was reported for the methanol-acetate combination (18). Conflicting reports ofthe simultaneous utilization of methanol-acetate(7, 8, 17) or H2-CO2 and acetate (7) by strains ofM. barkeri may be explained by (i) actual meta-bolic differences between various aceticlasticstrains or (ii) inadequate temporal observation ofthe initiation of methane formation without useof radioactive label. Real metabolic strain differ-ences for utilization of these substrate combina-tions have not been reported. However, thesimultaneous utilization of methanol and acetateby M. barkeri strain MS (17) may be explainedby inadequate observation of the origin of meth-ane; Scherer and Sahm (17) measured only totalgas production (CH4 and C02), and no radioac-tive substrates were employed. Similarly, themethanol-acetate studies of Hutten et al. (7)were based on gas production and not on specif-ic measurement of radioactively labeled CH4

0

0

E

0c

2

106 aa

S

C6a

S

2of

L-j

2 6 10 14 16 22

Time (days)FIG. 4. M. mazei growing in complex 0.05 M sodi-

um acetate medium with 5 RCi of 14CH3COO-, gassedwith N2-CO2 (70:30; 250 KPa) as indicated by thearrow. Symbols: 0, CH4; E, 14CH4.

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TABLE 2. Total radioactivity of spent cultures of Methanosarcina sp. strain 227 after acidification

Substrate(s) Label Headspace dpm' 14CH/14CO2 %14CH4 14co2 ratio Recoveryb

Acetate plus H2-CO2 14CH3COOH H2-CO2 plus N2 2.1 x 107 5.4 x 105 39 97Acetate 14CH3COOH N2 2.3 x 107 3.7 x 105 62 105Acetate plus H2-CO2 Na214CO3 H2-CO2 plus N2 2.8 x 106 1.9 x 106 1.5 85Acetate Na214CO3 N2 6.4 x 104 4.6 x 106 0.014 84

a Disintegrations per minute calculations include correction for soluble gases remaining in culture fluid.b Recovery was calculated as 100 [(disintegrations per minute recovered in gases)/(disintegrations per minute

added)].

and CO2. Hence, no conclusions can be madefrom these experiments regarding any short-term regulatory effects of H2-C02 or methanolon acetate catabolism to CH4 and CO2. Blautand Gottschalk (4) recently reported biphasicgrowth of M. barkeri strain Fusaro in whichtrimethylamine was preferentially metabolizedbefore acetate.Growth yields for strain 227 in complex medi-

um containing H2-CO2 were similar to thosereported for strain MS in defined medium (24),but higher than those reported for non-aceticlas-tic methanogens (16, 22). The higher H2-CO2growth yields for aceticlastic versus non-aceti-clastic methanogens may be due to differences ingrowth conditions as well as physiological char-acteristics. Hutten et al. (7) reported growthyields on methanol and acetate for strains MS,but YCH4 was not determined for H2-C02.Growth yields previously reported for strain 227in complex medium on methanol and acetate(18) are compared with the H2-CO2 yield inTable 4. The growth yield for each substrate wasdivided by the change in free energy for thatreaction as shown in Table 4 (YCH4/AG0'). Thisratio showed that the energy formed per mole-cule ofCH4 produced was similar for each of thethree substrates.Growth of strain 227 on acetate of H2-C02 in

mineral medium was characteristically muchslower than in complex medium. In addition,strain 227 could not be successfully transferredfrom mineral acetate medium to mineral H2-CO2medium, probably because of some nutritionalrequirement as previously suggested by L. Bar-esi (Dr.P.H. thesis, University of California,

Los Angeles, 1978). To initiate growth, it wasnecessary to use an inoculum of cells pregrownon H2-CO2 in complex medium.The nutritional status of the cells is reflected

in the differences in growth yields in mineralversus complex media. The average yield forstrain 227 grown on H2-CO2 in mineral mediumwas 5.4 to 6.4 mg/mmol of methane, whereas incomplex medium it was 8.4 mg/mmol of methaneproduced. Hutten et al. (7) reported a 1.5-foldincrease in growth yield (acetate or methanol)for strain MS grown in complex versus definedmedia. Lower growth yields and longer genera-tion times in mineral versus complex mediacorresponded to expected differences based onthe availability of assimilable carbon precursors.Growth conditions for the inoculum were im-

portant for determining the subsequent acetate-utilizing ability of Methanosarcina sp. strain227. Three or four transfers of the inoculum incomplex medium containing H2-CO2, but noacetate, resulted in diminished ability to useacetate immediately as the sole methanogenicsubstrate. However, three or four transfers ofthe inoculum in complex medium containing Hz-CO2 alone followed by transfer to a complex H2-CO2 medium containing acetate showed no lagin acetate utilization upon exhaustion of H2.Subsequent transfer of such a Hz-CO--acetate-grown culture to medium containing acetate asthe sole methanogenic substrate also resulted inimmediate growth. Winter and Wolfe also ob-served that pregrowth conditions affected subse-quent acetate-utilizing ability for M. barkeristrain MS (26). It is possible that synthesis ofintermediates necessary for utilization of acetate

TABLE 3. Methanol plus H2-CO2 mixed substrate experiment with Methanosarcina sp. strain 227

Substrates Label dpm 14CH4/14C02 %14CH4 14CO2 ratio Recovery'

Methanol plus N2-C02 14CH30H 1.33 x 106 5.32 x 105 2.50 89Methanol plus H2-C02 14CH3OH 1.55 x 106 2.76 x 105 5.63 87Methanol plus H2-C02 Na214CO3 1.24 x 106 9.35 x 106 0.133 95Hz-C02 Na214CO3 4.04 x 106 1.08 x 107 0.374 134

a See footnote b of Table 2.

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METHANOSARCINA METABOLISM OF MIXED SUBSTRATES 353

TABLE 4. Comparison of growth yields and changes in free energy for methanogenic reactions withMethanosarcina sp. strain 227

Reaction ~~~~AGO' YCH4 YCH4/AG0'Reaction (kJ/mol of CH)a (Mg/MMOIb ratio (gWkJ)CH3COO- + H20 -* CH4 + HC03- -31.0 2.1 -0.0684CH30H -. 3CH4 + HC03 + H+ + H20 -105.9 5.1 -0.048HCO3 + H+ + 4H2 - CH4 + 3H20 -135.1 8.4 -0.062

aDetermined from tables (25°C) compiled by Thauer et al. (23).b The data for acetate and methanol yields are from Smith and Mah (18).

may take place when H2-CO2 and acetate arepresent together, perhaps by synthesis of re-quired enzymes or cofactors by energy suppliedfrom H2 oxidation. Such biosynthetic require-ments may not be immediately met when H2-C02-grown cells are transferred to medium con-taining acetate as the sole methanogenic sub-strate. These results indicate that growth ofstrain 227 on H2-CO2 as the sole methanogenicsubstrate may select for a cell population withlimited acetate-utilizing ability. These findingscould also reflect genetic differences within theH2-CO2-grown population. The isolation of aMethanosarcina species incapable of H2-CO2utilization (28) and the results reported here forM. mazei indicate the actual existence of geneticdifferences among morphologically similarmethanogens. McInerney and Bryant (13) re-ported a similar phenomenon for strain MSgrown in yeast extract medium versus rumenfluid medium.

Zeikus et al. reported that M. barkeri andMethanobacterium thermoautotrophicum wereunable to "ferment" acetate in the absence ofhydrogen (27). In the presence of hydrogen,very small quantities of methane were producedby the reduction of both methyl and carboxylcarbons of acetate. Consequently, they pro-posed the following reaction for the origin ofmethane from acetate: H+ + CH3COO + 4H2-- 2CH4 + 2H20. This reaction is thermody-namically favorable and has a AG(' of about-83.6 kJ/mol of CH4; however, it is not asfavorable as the reduction of CO2 with H2 (Table4). In any case, the reduction of acetate in thismanner (27) is contrary to that reported by otherworkers (3, 5, 18, 19) and is probably not ofconsequence in the net formation of methanefrom acetate. We did observe a slight increase inthe oxidation of the methyl group of acetate inthe presence of H2-CO2 (Table 2). An increasein methyl group oxidation was also reported byWeimer and Zeikus for strain MS grown in thepresence of H2 (24, 25). Oxidation of the methylgroup of acetate increased for acetate-growncells in the presence of H2-CO2.Growth and methanogenesis from acetate by

strain 227 occurred without the addition of H2.Furthermore, very little, if any, added 14CO2

was reduced to methane in the absence of H2.These results are similar to those previouslyreported for strain 227 growing on acetate andmethanol (18). Strain 227 did not produce signifi-cant quantities of methane from acetate in thepresence of H2. The transition from H2-CO2 toacetate utilization occurred very rapidly, per-haps in response to the changing partial pressureof H2. This shift in methanogenesis from H2-CO2 to acetate was reflected in the changingspecific activity of methane (Fig. 1) derived frommethyl-labeled acetate. The initial radioactivityin methane may be attributed to utilization of asmall amount of the acetate (less than 0.2% ofthe total acetate pool) by residual enzymes car-ried over when the cells (pregrown on acetate)were transferred to the medium. When H2-CO2was added, the specific activity of CH4 fellbecause unlabeled CH4 was formed from theunlabeled H2-CO2, thereby diluting the 14CH4.After the exhaustion of H2-CO2 (98 to 99 h), themethanogenic aceticlastic reaction resumed, andthe specific activity of methane increased (Fig.1). A similar phenomenon, i.e., a decrease inacetate utilization in the presence of H2, wasrecently reported for M. barkeri strain MS (13).Acetate dissimilation in acetate-using cultures ofstrain MS did not increase until H2 wasexhausted. These results are in apparent con-trast to those reported by Hutten et al. (7), whoobserved acetate utilization in the presence ofH2-CO2 for strain MS, but no acetate utilizationin the presence of H2 alone. These differencesmay be due to the use of stationary incubationconditions which resulted in H2 limitation to thecells because of the low solubility of H2. Thishypothesis is supported by the reported lowrates of growth and methanogenesis from H2-CO2 (7) when strain MS was incubated withoutshaking (less than one-half of the rates on ace-tate). The reported requirement for CO2 (7) torelieve the inhibition by H2 may be due to H2removal by reduction of available CO2 to formCH4. Our data show that the aceticlastic reac-tion in M. mazei, which metabolized H2 slowly ifat all, remained inhibited even in the presence ofCO2. Thus, the role proposed by Hutten et al.for CO2 as an essential carbon supply for growthand methanogenesis may be secondary to its

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354 FERGUSON AND MAH

role as an oxidant for removal of H2.Although diauxic growth occurred for strain

227 on acetate and H2-CO2 mixtures, the cur-rent results do not support the hypothesis thatcatabolite repression was responsible (18), sincea shift to acetate metabolism occurred before thecomplete exhaustion of H2. A low H2 concentra-tion also accompanied the shift from H2 oxida-tion to acetate utilization in M. barkeri strain MS(13). Methanogenesis from acetate may be initi-ated because of diminished availability of H2 forCO2 reduction or because H2 is not an effectiveregulator at low concentration.

Isolation of a Methanosarcina species capableof methanogenesis from acetate but not H2-CO2is compatible with the hypothesis that two cellpopulations may exist, one capable of metabo-lizing only acetate and the other capable ofmetabolizing H2-CO2 (28). Likewise, the pres-ent results regarding H2-CO2 and acetate utiliza-tion for M. mazei support the proposal thatheritable metabolic differences do exist amongthe aceticlastic methanogens.

Results of the methanol-H2-CO2 experimentsindicated that methanogenesis from these twosubstrates was not sequential and that methanolreduction was stimulated by the addition of H2.Methanol reduction by H2 was also reported fora thermophilic strain of Methanosarcina sp. (11,28) as well as M. barkeri strain MS (24), and thispathway may be common for other strains ofMethanosarcina spp. Methanol was apparentlythe preferred electron acceptor when methanoland CO2 were both available for H2 oxidation togenerate energy for cellular metabolic needs.Under these conditions, reductants generatedfrom methanol oxidation for anabolic reactionswere also decreased (11), since methanol wasmainly reduced to methane. A coccus recentlyisolated by Miller and Wolin (14) grew only inthe presence of both H2 and methanol, but notwith either alone.

ACKNOWLEDGMENTSWe thank Arthur Heath, Adam Ng, Steven Dastic, and

Mario Panaqua for technical assistance and Michael Smith,Steven Zinder, Larry Baresi, Robert Sleat, Indra Mathrani,Harvey Negoro, Yitai Liu, and Danny Wong for suggestionsand discussions of our work.

This research was initially supported by Southern CaliforniaEdison Company through research grant 4-445930-2 and byPublic Health Service grant BRSG SO 7 RR5442 from theNational Institutes of Health. The final phase of this work wassupported by research grant DE-AT03-80ER10684 from theU.S. Department of Energy.

LITERATURE CITED1. Balch, W. E., and R. S. Wolfe. 1976. New approach to the

cultivation of methanogenic bacteria: 2-mercaptoethanol(HS-CoM)-dependent growth of Methanobacterium ru-minantium in a pressurized atmosphere. AppI. Environ.Microbiol. 32:781-791.

2. Baresi, L., R. A. Mah, D. M. Ward, and I. R. Kaplan.

1978. Methanogenesis from acetate: enrichment studies.Appl. Environ. Microbiol. 36:186-197.

3. Barker, H. A. 1943. Studies on the methane fermentation.VI. The influence of carbon dioxide concentration on therate of carbon dioxide reduction by hydrogen. Proc. Natl.Acad. Sci. U.S.A. 29:184-190.

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